Figure 2.

FACS screening of the MC4100A::Tn5 (pASKCB1-GFP) library and identification of the dnaJ::Tn5 transposon insertion that confers a large enhancement in CB1-GFP fluorescence and in the production of membrane-integrated CB1, as well as suppression of the cytotoxicity that accompanies CB1 expression. A: Enrichment of the MC4100A::Tn5 (pASKCB1-GFP) library with higher-fluorescence clones after repeated rounds of FACS screening. A fourfold increase in the average fluorescence (M) of the population was observed after two rounds of FACS sorting, and an 8-fold increase in CB1-GFP fluorescence was observed at saturation. B: Comparison of the CB1-GFP fluorescence of the parental MC4100A cells, the isolated strain GS101 (MC4100A dnaJ::Tn5), and of GS101 cells complemented with overexpressed dnaJ from the plasmid pBAD-DnaJ. C: Cell density at saturation of MC4100A and GS101 cells expressing CB1-GFP. The reported values correspond to the average of four replica experiments and the error bars represent one standard deviation from the mean value. OD₆₀₀: optical density at 600 nm. D: Western blots on isolated total membrane fractions of MC4100A and GS101 cells expressing CB1-GFP (left) and unfused CB1 (right) probed with an anti-polyhistidine antibody. The amount of membrane proteins loaded on each pair of lanes corresponds to an equal number of cells. No additional (degradation) bands were visible on the Western blots for GFP-free CB1. In all panels shown above, CB1-GFP and CB1 were expressed at 12°C for approximately 30 h. Fluorescence histograms correspond to a population of 10,000 cells. a.u: arbitrary units. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]